Food companies are required to test for presence of common food pathogens such as E. coli 0157:H7, Listeria, Salmonella, Campylobacter, Vibrio, etc. to protecting public health. Tests for detecting food pathogens are also performed by health service labs and government agencies to monitor and track outbreaks of food poisoning. Reducing the time necessary to obtain results of food testing is important for the food processing industry as it reduces the time and costs associated with storage of food prior to delivery till food testing results are obtained.
The most common method for identifying the presence of microorganisms is by enriching in selective broths and platting on defined agars. Classical platting methods require 3-5 days for confirmation (sometimes longer depending on the organism), and advanced skills in microbiology. The largest unmet need within the food testing market is the ability to produce results in one work shift, which is typically defined as eight hours or less.
Molecular methods such as PCR currently play a small role in testing presence of microorganisms. Real-time PCR is valuable because it combines simplicity with specificity and sensitivity. PCR, however, has its limitations, due to the necessity of sample preparation which can be time-consuming, and is technically challenging and expensive in view of the existence of a wide variety of food samples having different chemical and physical properties, and the necessity to process very large sample volumes. In addition many foods, such as meat products, contain PCR inhibitors.
One solution for this problem relies on magnetic properties. For example, magnetic seeds (magnetite) were used to capture single cell organisms in the presence of a calcium chloride binder, as described in U.S. Pat. No. 4,001,197. The magnetic beads can be DYNABEADS (Dynal AS, Oslo, Norway) functionalized to have positively or negatively charged, hydrophilic or hydrophobic surfaces, as taught in U.S. Pat. No. 7,560,228. Pathogen cells can also be captured by non-specific adsorption on the surfaces of magnetic beads (BUGS'n BEADS™ from NorDiag Inc., West Chester, Pa., US).
The above mentioned techniques rely on non-specific adsorption that may also bind various types of proteins that can inhibit and/or interfere with subsequent PCR reactions. The inhibition/interference can be exacerbated by subsequent in situ lysis and PCR in the presence of these proteins-bound magnetic beads.
Bacterial cellular surfaces comprise a variety of complex carbohydrate structures, such as glycoproteins, glycolipids, glycosaminoglycans, and proteoglycans. These glycoconjugates play a central role in cell-to-cell adhesion and subsequent recognition and receptor activation, as discussed in G. M. Whitesides et al., Angew. Chem. Int. Ed. 1998, 37, 2754-2794. And yet, the surfaces of different bacterial species are chemically and morphologically quite distinct.
Certain cells are able to selectively bind to one particular glycoconjugate but not the others. In practical applications, pathogen cells can be captured with magnetic beads having carbohydrates, including monosaccharides, disaccharides, oligosaccharides, and polysaccharides, immobilized on the bead surfaces, as described in the published U.S. Patent Application No. U.S. 2009/0186346A1. The binding of a pathogen cell onto a carbohydrate-modified magnetic bead is “monovalent” as schematically shown on FIG. 1.
The affinity, efficiency and binding strength of the structure depicted on FIG. 1 are weak. It may not withstand repetitive washing and rinsing to remove of debris and undesirable materials from the biological sample. Accordingly, better methods have to be employed to allow those skilled in the art to solve one or more of the above-mentioned problems.